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Article MEG3 Promotes Differentiation of Porcine Satellite Cells by Sponging miR-423-5p to Relieve Inhibiting Effect on SRF

Xiaofang Cheng 1 , Long Li 1, Gaoli Shi 1, Lin Chen 1, Chengchi Fang 1, Mengxun Li 1 and Changchun Li 1,2,* 1 A Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of the Ministry of Education and Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China; [email protected] (X.C.); [email protected] (L.L.); [email protected] (G.S.); [email protected] (L.C.); [email protected] (C.F.); [email protected] (M.L.) 2 The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China * Correspondence: [email protected]; Tel.: +86-158-7138-2527; Fax: +86-27-8728-0408



Received: 29 December 2019; Accepted: 13 February 2020; Published: 15 February 2020


Abstract: Although thousands of long noncoding RNAs (lncRNAs) have been identified in porcine growth and development, the regulation mechanisms of functional lncRNAs have not been well explored. In this study, using 50- and 30-rapid amplification of cDNA ends (RACE) assays, we obtained two different variants of lncRNA maternally expressed gene 3 (MEG3), namely, MEG3 v1 and MEG3 v2, that were both highly expressed in porcine skeletal muscle and in the early stage of the differentiation of porcine satellite cells. Moreover, we identified the core transcript MEG3 v2. Functional analyses showed that MEG3 overexpression could effectively arrest myoblasts in the G1 phase, inhibit DNA replication, and promote myoblast differentiation, whereas MEG3 knockdown resulted in the opposite effects. Interestingly, the expression of serum response factor (SRF), a crucial transcription factor for myogenesis process, remarkably increased and decreased in mRNA and protein levels with the respective overexpression and knockdown of MEG3. Dual luciferase reporter assay showed that MEG3 could attenuate the decrease of luciferase activity of SRF induced by miR-423-5p in a dose-dependent manner. MEG3 overexpression could relieve the inhibitory effect on SRF and myoblast differentiation induced by miR-423-5p. In addition, results of RNA immunoprecipitation analysis suggested that MEG3 could act as a ceRNA for miR-423-5p. Our findings initially established a novel connection among MEG3, miR-423-5p, and SRF in porcine satellite cell differentiation. This novel role of MEG3 may shed new light on understanding of molecular regulation of lncRNA in porcine myogenesis.

Keywords: porcine satellite cells; myoblast differentiation; MEG3; ceRNA; miR-423-5p

1. Introduction In mammalian genomes, only 2% of transcripts are translated into proteins. The vast majority of transcripts are noncoding RNAs, including microRNAs (miRNAs), Piwi-interacting RNAs, circular RNAs, and long noncoding RNAs (lncRNAs) [1]. Recently, an increasing number of researchers have focused on lncRNAs, which are a type of RNA with lengths of more than 200 nt and lack protein-coding potential [2]. They are characterized by less abundant, less evolutionarily conserved, and spatio-temporal specific expression profiles [3–5]. Thousands of functional lncRNAs have been identified to be involved in multiple biological processes, such as X chromosome inactivation,

Cells 2020, 9, 449; doi:10.3390/cells9020449 www.mdpi.com/journal/cells Cells 2020, 9, 449 2 of 20 genomic imprinting, stem cell maintenance, embryonic development, myogenesis, immunity, and tumorigenesis [6–11]. Mammalian skeletal muscle development is a complex process, which includes the following phases: somite commitment into progenitors, myoblast proliferation, migration, fusion, and final adaptation into fast-twitch and slow-twitch muscle fibers [12]. These development phases are regulated by the cascade control of multiple transcription factors, including myogenic regulatory factor 4 (MRF4), myogenic differentiation (MyoD), muscle bHLH proteins, Myf5, myogenin (MyoG), and myocyte enhancer factor 2 (MEF2) family [13–15]. A small number of lncRNAs, such as lncRNA Dum [16], MUNC [17,18], Linc-MD1 [19], Lnc-mg [20,21], LncMyoD [22], Linc-RAM [23], Linc-YY1 [24], SYISL [25], and lncRNA Irm [26], have been characterized to mediate biological processes of myoblasts during skeletal muscle development. These lncRNAs regulate skeletal muscle myogenesis and regeneration through various mechanisms, including chromosome modification, transcription activation, molecular sponge activity, competitive binding, mRNA translation, and protein stability. The lncRNA maternally expressed gene 3 (MEG3) on chromosome 7 is a conserved lncRNA among pig, humans, mice, and cow [22,27,28]. MEG3 is abundantly expressed in the paraxial mesoderm and is involved in callipyge phenotype of sheep, causing significant changes in muscle development and low fat content in the waist and hindquarters [29–33]. The knockout of MEG3 leads to skeletal muscle developmental defects and perinatal death [34]. In Qinchuan cattle, lncRNA MEG3 serves as a molecular sponge of miR-135, attenuating the suppressive effects of miR-135 upon MEF2C and thereby promoting skeletal differentiation [35]. Wang et al. proposed the potential roles of the MEF2A–MEG3/DIO3–PP2A signaling regulatory axis in bovine myoblast differentiation [36]. Downregulation of MEG3 can promote the proliferation and migration of smooth muscle cells of human pulmonary arteries [37]. In pigs, MEG3 has high expression levels in prenatal and early postnatal skeletal muscle [38], and four single nucleotide polymorphisms of MEG3 identified from Large White pigs are associated with meat-producing traits [39]. These findings indicated that MEG3 may be involved in myogenesis and contribute to skeletal muscle development in pigs. However, the molecular mechanisms need to be further explored. To verify our speculation, we further detected the expression profile of MEG3 and explored its function and molecule mechanism in the myogenesis of porcine satellite cells. In this study, we observed that the expression level of MEG3 in skeletal muscle was higher than that in other tissues, and MEG3 was differentially expressed in the myogenesis of porcine satellite cells. Moreover, MEG3 could effectively inhibit myoblast proliferation and promote myoblast differentiation. Mechanistically, MEG3, as a competing endogenous RNA (ceRNA), promotes the differentiation of porcine satellite cells by sponging miR-423-5p to relieve the inhibiting effect on serum response factor (SRF). Our results may contribute to a better understanding of the lncRNA–miRNA–target-gene regulatory network in the differentiation of porcine satellite cells.

2. Materials and Methods

2.1. Animals Animals used in this study were <7-days-old male Large White piglets. Six piglets were euthanized, and organs and tissues were collected after dissection for tissue expression profile analysis. For porcine satellite cells isolation, the hind leg muscles from three other piglets were rapidly pooled, minced and digested. Animals care and experimentation in this study have been performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and have been approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, Wuhan, China (permit HZAUSW2015-0003). Cells 2020, 9, 449 3 of 20

2.2. Cell Culture Satellite cells were primarily isolated from hind leg muscles of <1-week-old male piglets. Briefly, skeletal muscles were minced into pieces and digested with 300 U/mL type II collagenase (Gibco; Gaithersburg, MD, USA) in a shaking water bath at 37 ◦C for 2.5 h. After having been terminated with high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), the cell suspension was filtered through 100, 70, and 40 µm filters to remove tissue debris. Afterwards, the cell pellet was ultimately resuspended and cultured in RPMI-1640 medium supplemented with 20% FBS (Gibco), 0.5% Chicken Embryo Extract (Gemini, Woodland, CA, USA), 1% GlutaMax (Gibco), 1% NEAA (Gibco), 1% Anti-Anti (Gibco), 2.5 µg/L basic fibroblast growth factor (bFGF; Invitrogen, Grand Island, NY, USA). Due to differential adhesion property, fibroblasts among the mixed cells were removed after being incubated in uncoated plates for 2.5 h. The purified satellite cells were transferred into the Matrigel (BD Biosciences, CA, USA) coated plates for proliferation cultures. When porcine satellite cells were grown to 90% confluence, they were transferred into DMEM supplemented with 5% Horse Serum (HS; Gibco) to induce differentiation. The PK15 cells were cultured in DMEM supplemented with 10% FBS (Gibco). All cells described above were incubated at 37 ◦C in 5% CO2.

2.3. RNA Oligonucleotide and Plasmid Construction The miR-423-5p mimic, mimic negative control (NC) and the antisense oligonucleotide (ASO) oligo against MEG3 and scrambled oligo were purchased from RiboBio (Guangzhou, China). Small interfering RNA (siRNA) of SRF and scrambled oligo were designed and synthesized from GenePharma (Shanghai, China). Oligonucleotide sequences in this study are shown in Table S1. For the overexpression plasmids, the full lengths of two different transcripts of porcine MEG3 gene were synthesized from Tsingke (Beijing, China) and cloned into the pZW1-son plasmid. The coding DNA sequence (CDS) of SRF was amplified by PCR and cloned into the pcDNA3.1 plasmid. The major primers used in this study are listed in Table S2. For the dual-luciferase reporter vector plasmid construction, about 300 bp wild-type and mutant sequences of MEG3 and SRF, containing miR-423-5p seed sequence target sites, were inserted into pGL3-Basic vector. Mutant plasmid of MEG3 was generated by changing the binding site of miR-423-5p from CTGCCCCT to GACGATAG; that of SRF was changed from CTGCCCCTCA to GACGGAGTAT. All the recombinant plasmids were confirmed by sequencing (Sangon Biotech, Shanghai, China).

2.4. Cell Transfection All transient transfections in porcine satellite cells or PK15 cells were performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

2.5. Nuclear and Cytoplasmic RNA Fractionation Cells were prepared at both proliferative and differentiated periods. The procedure for separating the nuclear and cytoplasmic RNA fractionation was performed in accordance with previous published reports [40]. RNAs of cytoplasmic and nuclear fractions were extracted with RNAiso reagent (TaKaRa, Otsu, Japan). The locations of MEG3 (detection with the overlap region primers of MEG3 two transcripts), GAPDH (cytoplasmic marker gene) and Neat1 (a nuclear expression lncRNA) were analyzed by quantitative polymerase chain reaction (qPCR). Primer sequences for qPCR are listed in Table S2.

2.6. RNA Extraction, cDNA Synthesis, and Quantitative Polymerase Chain Reaction(qPCR) Total RNA was extracted from cells using RNAiso reagent (TaKaRa, Otsu, Japan) according to the manufacturer’s instructions. The concentration and quality were measured by a spectrophotometer Cells 2020, 9, 449 4 of 20

(Nanodrop 2000, Thermo Fisher Scientific, Wilmington, DE, USA) at 260 and 280 nm. Ratios of absorption (260/280 nm) of all samples ranged from 1.8 to 2.0. Complementary DNA (cDNA) synthesis for messenger RNA (mRNA) was performed using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Otsu, Japan). For miRNA, stem loop miRNA qRT-PCR primers specific for miR-423-5p and U6 were designed by Vazyme (Nanjing, China) and cDNA was synthesized with miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, Nanjing, China). Quantitative polymerase chain reaction (qPCR) for mRNA was carried out on a Bio-Rad CFX96 Real-Time Detection System using TB Green Premix Ex Taq II (Tli RNase H Plus) (TaKaRa, Otsu, Japan). For miRNA, miRNA Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used ∆∆CT and analyzed with the 2− method. All primers for qPCR in this study were designed with Primer 5, and primer sequences are listed in Table S2. The 18S ribosomal RNA (18S rRNA) and U6 were used as internal controls.

2.7. 50 and 30 RACE and Full-Length LncRNA Cloning To obtain the transcription information and full-length sequences of MEG3, SMARTer RACE cDNA Amplification Kit (Clontech, Osaka, Japan) was used for 50 and 30 RACE according to the manufacturer’s instructions. The gene-specific primers (GSP) for RACE PCR were designed for 50 RACE (GSP1) and 30 RACE (GSP2) amplification. The PCR products were inserted into the pRACE vector and sequenced by Tsingke Biological Technology (Wuhan, China). The gene-specific primers are shown in Table S2.

2.8. CCK-8 Assay Porcine satellite cells were seeded in 96-well plates and transfected with ASO oligo against MEG3 or MEG3 overexpression vectors when cell confluence was 40–50%. Then, cell proliferation was monitored with the CCK-8 Cell Counting Kit (Vazyme, Nanjing, China) following the manufacturer’s protocol. And the absorbance at 450 nm was measured in the spectrophotometer after being transfected for 12, 24, 36, and 48 h.

2.9. 5-Ethynyl-20-deoxyuridine (EdU) Assay Porcine satellite cells were transferred to culture medium with 50 µM EdU (RiboBio, Guangzhou, China) for 2 h at 37 ◦C after 36 h transfection. Afterwards, cells were fixed in 4% paraformaldehyde for 15 min at room temperature (RT), and then permeabilized with 0.3% Triton X-100 for 10 min. To block unspecific binding, cells were incubated in the blocking buffer (PBS containing 3% bovine serum albumin, 0.3% Triton X-100) for 1 h at RT. Then cells were incubated with a solution containing 10 mM EdU in dark for 30 min. The nuclei were stained with 10 µg/mL 4, 6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) solution in dark for 10 min. Leica SP8 confocal microscope was used to capture three randomly selected fields to visualize the number of EdU-stained cells.

2.10. Flow Cytometry Analysis For flow cytometry analysis of cell cycle, the Cell Cycle Detection Kit (Keygen, Nanjing, China) were used in line with the manufacturer’s instructions. Briefly, cells were harvested and fixed in 70% ethanol overnight at 4 ◦C after 36 h transfection. Then cells were rinsed with PBS and centrifuged at 2500 rpm for 5 min. Subsequently, cells were stained with prepared propidium iodide (PI) solution, containing RNase A and PI at a volume ratio of 1:9, and then incubated in dark for 30 min at RT. Flow cytometry analysis was performed on Beckman Coulter FC500 Cytometer (Beckman Coulter, Miami, FL, USA) and data were processed by ModFit software (Verity Software House, Topsham, ME, USA). Cells 2020, 9, 449 5 of 20

2.11. Transcriptome Sequencing and Differential Expression Analysis To further study the involvement of MEG3 in porcine myogenesis, we thoroughly analyzed RNA-seq data from MEG3 knockdown and control groups in porcine satellite cells differentiated for 30 and 40 h. In total, 3 µg of RNA for each sample was used to construct sequencing libraries. The libraries were sequenced on the Illumina HiSeq X-ten platform and 150 bp paired-end reads were generated. Then, we used FastQC software (Nanjing Agricultural University, Nanjing, China) to evaluate the quality of obtained sequence data and used Trimmomatic tool (version 0.3.2, Nanjing Agricultural University, Nanjing, China) to trim. Next, HISAT2 (version 2.0.1, Iowa State University, Ames, IA, USA) was used to obtain the qualified and clean reads mapped to the pig reference genome (Sus scrofa 11.1) and StringTie (version 1.3.4, Johns Hopkins University, Baltimore, MD, USA) was used to assemble the mapped reads with default parameters. HTSeq-count (version 0.9.1, European Molecular Biology Laboratory, Heidelberg, BW, Germany) was used to count reads mapped to the genome and the annotation file. Subsequently, differentially expressed genes were identified utilizing the R packages DESeq2 (Tsinghua University, Beijing, China). A transcript will be considered as differentially expressed between two groups if the absolute value of log2 (fold-change) > 1, p-value < 0.05 and false discovery rate (FDR) < 0.05. In order to query each protein-coding gene and understand their functions, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis by running queries for each protein-coding gene against the DAVID database. GO terms or KEGG pathways with corrected p-value < 0.05 were considered to be enriched clusters. Because of the limitation of genes annotation in Sus scrofa, all genes were converted into human homologous genes using BIOMART from Ensembl.

2.12. Dual-Luciferase Reporter Assay When cell confluence reached about 80%, the wild-type and mutant dual-luciferase reporter vectors of MEG3 or SRF were separately co-transfected into PK15 cells with pRL-TK normalizing reporter plasmid and miR-423-5p mimic. For the interaction among MEG3, miR-423-5p, and SRF, wild-type dual-luciferase reporter vectors of SRF, miR-423-5p mimic, and different doses of pcDNA3.1-MEG3 plasmids (0, 1, and 2 µg) were co-transfected into PK15 cells. After incubation for 48 h, the cells were harvested. The firefly and renilla luciferase activities were measured using Dual-Luciferase® Reporter Assay System (Promega, Fitchburg, WI, USA).

2.13. RNA Immunoprecipitation Assay MEG3 To identify specific RNA molecules associated with Argonaute2 (Ago2), a key effector of small RNA mediated gene silencing [41], we performed RNA immunoprecipitation (RIP) assay with EZ-Magna RIP Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Briefly, porcine satellite cells, differentiated for 48 h, were collected and lysed in RIP lysis buffer. Then, cell lysates were incubated with A/G magnetic beads conjugated with anti-Ago2 antibody (Boster, Wuhan, China). Then, the immunoprecipitated RNA was isolated and qPCR was performed to detect the abundance of MEG3, SRF, and miR-423-5p; 18S rRNA and U6 were used as internal controls. Primer sequences for qPCR are listed in Table S2.

2.14. Immunofluorescence Staining Cells were fixed in 4% paraformaldehyde for 15 min after 48 h transfection and then permeabilized in 0.3% Triton X-100 for 10 min. Subsequently, cells were blocked with blocking solution (3% BSA, 0.3% Triton X-100, 10% FBS complemented with PBS) for 2 h. Then, immunofluorescence staining was performed using anti-MyHC (Millipore, Billerica, MA, USA; 1:1000) or anti-MyoG (Abcam, Cambridge, MA, USA; 1:500) overnight at 4 ◦C. After that, cells were stained with Alexa 594-labeled anti-mouse antibody (Antgene, Wuhan, China; 1:200) for 1 h. The cell nuclei were visualized using DAPI (Invitrogen, Carlsbad, CA, USA) solution in darkness for 10 min. Images from three randomly Cells 2020, 9, 449 6 of 20 selected fields were obtained with Leica SP8 confocal microscope and processed with Image J software (version 1.48, National Institutes of Health, Bethesda, MD, USA).

2.15. Western Blot Total protein was extracted from porcine satellite cells and lysed in RIPA lysis buffer with 1% PMSF. The protein concentration was detected with Pierce BCA Protein Assay Reagent (Thermo-Fisher, Waltham, MA, USA). Then, we performed immunoblotting with various antibodies according to standard procedures. The primary antibodies were diluted as follows: MyoG (Abcam, Cambridge, MA, USA) 1:1000, MyoD (Abclonal, Wuhan, China) 1:1000, MyHC (Millipore, Billerica, MA, USA) 1:3000, and β-tubulin (Servicebio, Wuhan, China) 1:2000. The horseradish peroxidase (HRP)-conjugated secondary antibodies (1:4000) (anti-rabbit IgG, or anti-mouse IgG; Servicebio, Wuhan, China) were used to detect protein expression.

2.16. Statistical Analysis Generally, results are presented as the means standard error of the mean (SEM). Statistical ± comparison between two different groups were assessed by two-tailed Student’s t-test. The p value < 0.05 was considered to be statistically significant.

3. Results

3.1. Expression of MEG3 LncRNA To investigate the regulatory mechanism in pigs, we first evaluated the full-length cDNA of MEG3 in porcine satellite cells using 50 and 30 RACE (Figure1A). We designed gene-specific primers (GSP) for RACE and identified two polyadenylated MEG3 transcripts: MEG3 variant 1 (MEG3 v1), 1430 nt in length, and MEG3 variant 2 (MEG3 v2), 1380 nt in length. Sequence analysis showed that these two different variants shared the same 508-nt front part (Figure1B). Interestingly, MEG3 showed remarkably higher mRNA expression levels in brain and muscle tissues, such as longissimus dorsi and gastrocnemius muscle, than other multiple tissues (Figure1C). Notably, the expression of MEG3 v2 showed a substantial dominance in porcine muscle tissues compared with MEG3 v1 (Figure1C). We collected porcine satellite cells at the proliferation stage every 6 h and at the differentiation stage every 12 h to distinguish the expression levels of these two MEG3 variants in different periods of myogenesis. Overall, two variants of MEG3 had similar expression trends. Both variants were upregulated in the proliferation and early stage of differentiation and reached their peaks in porcine satellite cells differentiated for 48 h, but they gradually decreased afterward (Figure1D), suggesting that MEG3 could be a promyogenic factor during the early differentiation in porcine satellite cells. Obviously, MEG3 v2 was the most abundant in porcine satellite cells with dominant expression level compared with that of MEG3 v1 (Figure1D). Therefore, MEG3 v2 was the core transcript in porcine satellite cells. Moreover, nuclear–cytoplasmic RNA fractionation experiments demonstrated that MEG3 was mainly located in the nuclear compartment of proliferating myoblasts (76.86%) and differentiated myotubes (59.99%). Interestingly, its proportion in the cytoplasm had increased in myotubes (in a range from 23.14% to 40.01%) (Figure1E). Cells 20202020,, 99,, 449x 77 of of 20

Figure 1. Characterization of the lncRNA MEG3 gene. (A) Results of 50 rapid amplification of cDNA endsFigure (RACE) 1. Characterization and 30 RACE of of the lncRNA lncRNAMEG3 MEG3in gene. porcine (A) skeletal Results muscle.of 5′ rapid The amplification arrows represent of cDNA the twoends transcripts (RACE) and variants 3′ RACE of MEG3 of lncRNA.(B) Structure MEG3 in of porcine two transcript skeletal variantsmuscle. The (MEG3 arrowsv1 and representMEG3 v2)the oftwo the transcripts porcine MEG3 variantsgene. of MEG3 The grey. (B) shades Structure mean of two that transcript the two transcripts variants (MEG3 sharethe v1 and same MEG3 front v2) parts. of GSP1the porcine and GSP2 MEG3 are gene. the gene-specific The grey shades primers mean for that 50 and the 3tw0 RACEo transcripts of MEG3 share.(C )the Expression same front levels parts. of theGSP1 two andMEG3 GSP2variants are the ingene-specific heart, spleen, primers lung, kidney,for 5′ and cerebrum, 3′ RACEcerebellum, of MEG3. (C longissimus) Expression dorsi levels and of gastrocnemius.the two MEG3 variants (D) Expression in heart, levels spleen, of the lung, two kidney,MEG3 variantscerebrum, during cerebellum, porcine longissimus myoblast proliferation dorsi and andgastrocnemius. differentiation. (D) ( EExpression) The distribution levels ofofMEG3 the coretwo variantMEG3 transcriptvariants (MEG3duringv2) porcine in the cytoplasmmyoblast andproliferation nuclei of and porcine differentiation. proliferous ( myoblastE) The distribution and myotube of MEG3 differentiated core variant for48 transcript h. NEAT1 (MEG3is a known v2) in nuclearthe cytoplasm lncRNA, and and nucleiGAPDH ofis porcine a cytoplasmic-enriched proliferous myoblast gene. The and relative myotube RNA differentiated levels were normalized for 48 h. to those of the control 18S rRNA. Error bars represent mean SEM of three biological replicates. NEAT1 is a known nuclear lncRNA, and GAPDH is a cytoplasmic-enriched± gene. The relative RNA Statisticallevels were significance normalized of to di ffthoseerences of the was control assessed 18S by rRNA Student’s. Errort-test. bars NC,represent negative mean control. ± SEM of three 3.2. MEG3biological Inhibits replicates. Myoblast Statistical Proliferation significance of differences was assessed by Student’s t-test. NC, negative control. MEG3 expression levels were upregulated during the myoblast proliferation phase, indicating that3.2. MEG3MEG3 Inhibitscould beMyoblast involved Proliferation in the regulation of myoblast proliferation. In functional deficit and acquisition experiments, optimized phosphorothioate-modified antisense oligodeoxynucleotide (ASO) MEG3 expression levels were upregulated during the myoblast proliferation phase, indicating against the overlapping region sequence of the two MEG3 variants and pZW1-MEG3 v1 or pZW1-MEG3 that MEG3 could be involved in the regulation of myoblast proliferation. In functional deficit and v2 plasmid were respectively transfected into porcine satellite cells. In the 5-ethynyl-2 -deoxyuridine acquisition experiments, optimized phosphorothioate-modified antisense oligodeoxynucleotide0 (EdU) staining assays, the interference of MEG3 with ASO showed higher mitotic activity with an (ASO) against the overlapping region sequence of the two MEG3 variants and pZW1-MEG3 v1 or increase in EdU incorporation (Figure2A). On the contrary, the overexpression of MEG3 v1 (p < 0.05) pZW1-MEG3 v2 plasmid were respectively transfected into porcine satellite cells. In the or MEG3 v2 (p < 0.01) showed lower mitotic activity with a decrease in EdU positivity (Figure2B). 5-ethynyl-2′-deoxyuridine (EdU) staining assays, the interference of MEG3 with ASO showed higher The CCK-8 assay showed that the MEG3 knockdown for 24, 36, or 48 h could dramatically accelerate mitotic activity with an increase in EdU incorporation (Figure 2A). On the contrary, the cellular proliferation (Figure2C). Inversely, the overexpression of MEG3 v1 or MEG3 v2 substantially overexpression of MEG3 v1 (p < 0.05) or MEG3 v2 (p < 0.01) showed lower mitotic activity with a suppressed the proliferative ability of porcine satellite cells compared with the negative control decrease in EdU positivity (Figure 2B). The CCK-8 assay showed that the MEG3 knockdown for 24,

Cells 2020, 9, x 8 of 20

Cells 2020, 9, 449 8 of 20 36, or 48 h could dramatically accelerate cellular proliferation (Figure 2C). Inversely, the overexpression of MEG3 v1 or MEG3 v2 substantially suppressed the proliferative ability of porcine (Figuresatellite2 cellsD). The compared propidium with iodide the negative flow cytometry control (Figure assays 2D). indicated The propidium a considerable iodide reduction flow cytometry of cell quantityassays indicated in the G0 a/ G1considerable phase and reduction a remarkable of cell increase quantity of cellin the quantity G0/G1 in phase the S phaseand a afterremarkableMEG3 knockdownincrease of (Figurecell quantity2E,F). Conversely,in the S phase the overexpression after MEG3 knockdown of MEG3 v1 or(FigureMEG3 2E,F).v2 showed Conversely, an opposite the eoverexpressionffect (Figure2G,H). of MEG3 These v1 findings or MEG3 validated v2 showed that anMEG3 oppositecould effect inhibit (Figure the proliferation 2G,H). These of findings porcine satellitevalidated cells. that MEG3 could inhibit the proliferation of porcine satellite cells.

Figure 2. MEG3 inhibits myoblast proliferation. (A,B) EdU staining assays after MEG3 knockdown (A) or overexpression of the two MEG3 variants (B) showing the EdU incorporation in proliferous Figure 2. MEG3 inhibits myoblast proliferation. (A,B) EdU staining assays after MEG3 knockdown porcine satellite cells. The S phase of mitosis cells were stained with EdU. The nuclei were stained (A) or overexpression of the two MEG3 variants (B) showing the EdU incorporation in proliferous with DAPI. Scale bar: 50 µm. (C,D) CCK-8 cell proliferation assays of porcine satellite cells suggested porcine satellite cells. The S phase of mitosis cells were stained with EdU. The nuclei were stained that MEG3 knockdown significantly promoted myoblast proliferation after transfection with antisense with DAPI. Scale bar: 50μm. (C,D) CCK-8 cell proliferation assays of porcine satellite cells suggested oligonucleotide (ASO)–MEG3 for 24, 36, and 48 h, compared with the negative control groups (C), that MEG3 knockdown significantly promoted myoblast proliferation after transfection with while overexpression of MEG3 v1 or MEG3 v2 inhibited myoblast proliferation (D). (E,G) Flow cytometry antisense oligonucleotide (ASO)–MEG3 for 24, 36, and 48 h, compared with the negative control analysis after MEG3 knockdown (E) or overexpression of the two MEG3 variants’ overexpression plasmidsgroups (C for), while 36 h ( Goverexpression). (F,H) Histogram of MEG3 bar graph v1 orsummarizing MEG3 v2 inhibited the flow myoblast cytometry proliferation analysis showing (D). (E, theG) numberFlow cytometry of cells in analysis each cell after cycle MEG3 phase. knockdown Statistical results (E) or showing overexpression that MEG3 of theknockdown two MEG3 significantly variants’ increasedoverexpression the percentage plasmids for of S-phase36 h (G). cells (F,H ()F Histogram), while MEG3 bar graphoverexpression summarizing significantly the flow cytometry decreased theanalysis percentage showing of the S-phase number cells of ( Hcells). Error in each bars ce representll cycle phase. mean StatisticalSEM of results three biologicalshowing that replicates. MEG3 ± Statisticalknockdown significance significantly of increased differences the was percentage assessed of by S-phase Student’s cellst-test. (F), while * p < MEG30.05, ** overexpressionp < 0.01. NC, negativesignificantly control. decreased the percentage of S-phase cells (H). Error bars represent mean ± SEM of three biological replicates. Statistical significance of differences was assessed by Student’s t-test. * p < 0.05, ** p < 0.01. NC, negative control.

Cells 2020, 9, 449 9 of 20

3.3. MEG3 Promotes Myoblast Differentiation The above results demonstrated that MEG3 is extremely important for myoblasts to be able to withdraw from the cell cycle, a crucial step in myoblast differentiation. In addition, the expression profile of MEG3 prompted its association with myoblast differentiation. We used qPCR, Western blot, and immunofluorescence staining to test the changes of three established myogenic marker genes (MyoD, MyoG, and myosin heavy chain (MyHC)). qPCR results showed that MEG3 was successfully knocked down in the ASO–MEG3 group myotube differentiated for 48 h (Figure3A). Meanwhile, the mRNA and protein levels of MyoD, MyoG, and MyHC were remarkably downregulated after MEG3 knockdown compared with the control group (Figure3A,B). Consistently, immunofluorescence staining of MyoG and MyHC showed that MEG3 knockdown notably reduced the proportion of MyoG+ and MyHC+ cells (Figure3C,D). To further confirm the above observation, we successfully overexpressed MEG3 v1 and MEG3 v2 in porcine satellite cells in the differentiation phase (Figure3E). As expected, the mRNA and protein levels of MyoD, MyoG, and MyHC substantially increased after MEG3 v1 and MEG3 v2 overexpression in myotube (Figure3F,G). Likewise, MEG3 overexpression showed that more cells proceeded with differentiation and myotube formation than the control group in the immunofluorescence staining of MyoG and MyHC (Figure3H,I). Taken together, our results confirmed that MEG3 is required for myoblast differentiation.

3.4. Gene Expression Profile of MEG3 Knockdown in Porcine Satellite Cells Tofurther study the involvement of MEG3 in skeletal muscle development, we thoroughly analyzed RNA-seq data from MEG3 knockdown and control groups in porcine satellite cells differentiated for 30 and 40 h and identified differentially expressed genes between the two groups. In total, we obtained over 40 million raw reads from each library. Then we removed low-quality sequences, and the clean reads mapped more than 95% of the raw data. Next, we aligned all clean reads to the porcine Sscrofa11.1 reference genome and found that more than 70% clean reads could be uniquely mapped to the genome (Table S3). The principal component analysis (PCA) score plots showed that datasets from four groups (30 hNC, 30 hASO and 40 hNC, 40 hASO groups for MEG3 knockdown) were clustered separately (Figure4A). Hierarchical clustering was performed for di fferential expressions of protein-coding genes and obtained a global overview of gene expression profile among 40 hNC group and 40 hNC ASO group for MEG3 knockdown (Figure4B). Finally, the di fferentially expressed genes were used to perform gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In detail, we identified 273 and 207 differentially expressed protein-coding genes from MEG3 knockdown for the 30 and 40 h groups, respectively, compared with the corresponding negative control groups. At 30 h comparison groups, GO enrichment analysis of these differentially expressed genes indicated that they were mainly associated with muscle-related processes, including muscle myosin complex, skeletal muscle contraction, and positive regulation of myoblast differentiation, skeletal muscle cell differentiation, and structural constituent of muscle (Figure4C). KEGG pathway enrichment analysis was used to explore the biological pathways for differentially expressed protein-coding genes. The results showed that these genes participated in the PI3K–Akt signaling pathway, oxytocin signaling pathway, focal adhesion, and adrenergic signaling in cardiomyocytes (Figure4D). The GO enrichment analysis at 40 h indicated that the differentially expressed genes were also related to muscle-related processes, such as Z disc, actin cytoskeleton, myofibril, muscle contraction, skeletal muscle cell differentiation, and structural constituent of muscle (Figure4E). KEGG pathway enrichment analysis showed that these differentially expressed genes were mainly involved in the PI3K–Akt signaling pathway, regulation of actin cytoskeleton, focal adhesion, and MAPK signaling pathway (Figure4F). Among these differentially expressed genes, CCND1 [42], PLCB1 [43], MEF2C [44], and FOXO3 [45,46] have a known function in regulating myogenesis. As a consequence, MEG3 could specifically have important effects on skeletal muscle development. To validate the reliability of our sequencing results, we randomly selected several differentially expressed genes in 30 and 40 h groups for qPCR to detect their expression levels. The expression levels of CAMK1, ITGA3, PLK2, CCND1, EIF4E, ITGA7, ITPR3, Cells 2020, 9, x 9 of 20

3.3. MEG3 Promotes Myoblast Differentiation The above results demonstrated that MEG3 is extremely important for myoblasts to be able to withdraw from the cell cycle, a crucial step in myoblast differentiation. In addition, the expression profile of MEG3 prompted its association with myoblast differentiation. We used qPCR, Western blot, and immunofluorescence staining to test the changes of three established myogenic marker genes (MyoD, MyoG, and myosin heavy chain (MyHC)). qPCR results showed that MEG3 was successfully knocked down in the ASO–MEG3 group myotube differentiated for 48 h (Figure 3A). Meanwhile, the mRNA and protein levels of MyoD, MyoG, and MyHC were remarkably downregulated after MEG3 knockdown compared with the control group (Figure 3A,B). Consistently, immunofluorescence staining of MyoG and MyHC showed that MEG3 knockdown notably reduced the proportion of MyoG+ and MyHC+ cells (Figure 3C,D). To further confirm the above observation, we successfully overexpressed MEG3 v1 and MEG3 v2 in porcine satellite cells in the differentiation phase (Figure 3E). As expected, the mRNA and protein levels of MyoD, MyoG, Cellsand2020 MyHC, 9, 449 substantially increased after MEG3 v1 and MEG3 v2 overexpression in myotube (Figure10 of 20 3F,G). Likewise, MEG3 overexpression showed that more cells proceeded with differentiation and myotube formation than the control group in the immunofluorescence staining of MyoG and MyHC and(FigurePLCB1 3H,I).were Taken upregulated, together, whereas our thoseresults of ACACBconfirmed, FOXO3 that , MEF2CMEG3 ,isMYL4 required, MYLK4 for, andmyoblastTPM2 weredifferentiation. downregulated (Figure4G,H).

Figure 3. MEG3 promotes myogenic differentiation of porcine satellite cells. (A,B) The real-time PCR (A) and Western blot analysis (B) showing that MEG3 knockdown significantly decreases expression levels of myogenic marker genes (MyoD, MyoG, MyHC) and SRF.(C,D) Immunofluorescence staining of MyoG (C) and MyHC (D) in porcine satellite cells after transfection with ASO–MEG3 or ASO–NC showing that MEG3 knockdown significantly reduces the positively stained cells of MyoG and MyHC. (E) The detection of overexpression efficiency of MEG3 v1 and MEG3 v2. (F–I) qPCR (F), Western blot (G), and immunofluorescence staining of MyoG (H) and MyHC (I) results showing that MEG3 overexpression promotes satellite cell differentiation. Scale bar of (C,H): 50 µm. Scale bar of (D,I): 100 µm. Porcine satellite cells were harvested after transfection with MEG3 ASO or overexpression plasmid and differentiation for 48 h. The relative mRNA levels were normalized to those of the control 18S rRNA. The relative protein levels were normalized to those of the control β-tubulin. Error bars represent mean SEM of three biological replicates. Statistical significance of differences was assessed ± by Student’s t-test. * p < 0.05, ** p < 0.01. N.S. means that there was no significant difference. NC, negative control. qPCR, quantitative polymerase chain reaction. Cells 2020, 9, 449 11 of 20 Cells 2020, 9, x 11 of 20

Figure 4. Effect of MEG3 downregulation on myogenesis transcriptome. (A) PCA analysis of four Figure 4. Effect of MEG3 downregulation on myogenesis transcriptome. (A) PCA analysis of four datasets (30 hNC, 30 hASO and 40 hNC, 40 hASO groups) for MEG3 knockdown. (B) Hierarchical datasets (30 hNC, 30 hASO and 40 hNC, 40 hASO groups) for MEG3 knockdown. (B) Hierarchical clustering of differentially expressed genes after transfection of porcine satellite cells with ASO–MEG3 clustering of differentially expressed genes after transfection of porcine satellite cells with or ASO–NC and differentiation for 40 h. The color-ratio bar at the right indicates intensity of ASO–MEG3 or ASO–NC and differentiation for 40 h. The color-ratio bar at the right indicates gene upregulation (blue) and downregulation (white). (C–F) GO enrichment and KEGG pathway intensity of gene upregulation (blue) and downregulation (white). (C–F) GO enrichment and KEGG scatterplot analysis for differentially expressed genes for cells differentiated for 30 (C,E) and 40 h (D,F). pathway scatterplot analysis for differentially expressed genes for cells differentiated for 30 (C,E) (G,H) Validation of differentially expressed genes involved in muscle development by qPCR. The y-axis and 40 h (D,F). (G,H) Validation of differentially expressed genes involved in muscle development indicates the fold change of RNA-Seq and qPCR. The relative mRNA levels were normalized to those by qPCR. The y-axis indicates the fold change of RNA-Seq and qPCR. The relative mRNA levels were of the control 18S rRNA. Error bars represent mean SEM of three biological replicates. Statistical normalized to those of the control 18S rRNA. Error bars± represent mean ± SEM of three biological significance of differences was assessed by Student’s t-test. ** p < 0.01. NC, negative control. ASO, replicates. Statistical significance of differences was assessed by Student’s t-test. ** p < 0.01. NC, antisense oligonucleotide. PCA, Principal component analysis. GO, Gene ontology. KEGG, Kyoto negative control. ASO, antisense oligonucleotide. PCA, Principal component analysis. GO, Gene Encyclopedia of Genes and Genomes. qPCR, quantitative polymerase chain reaction. ontology. KEGG, Kyoto Encyclopedia of Genes and Genomes. qPCR, quantitative polymerase chain 3.5.reaction. MEG3 Acts As a ceRNA for miR-423-5p Given that lncRNAs can regulate multiple biological functions by sponging regulatory miRNAs, 3.5. MEG3 Acts As a ceRNA for miR-423-5p the seed regions of miR-423-5p were predicted to be complementary with MEG3 core transcript MEG3 v2 (abbreviatedGiven that aslncRNAsMEG3), andcan anotherregulate potential multiple target biological gene (i.e., functionsSRF) of by miR-423-5p sponging was regulatory obtained miRNAs,using TargetScan the seed (regionshttp://www.targetscan.org of miR-423-5p were/vert_71 predicted/) and to RNAhybridbe complementary software with (https: MEG3//bibiserv. core transcriptcebitec.uni-bielefeld.de MEG3 v2 (abbreviated/rnahybrid /)as (Figure MEG35A).), andSRF anotheris a crucial potential transcription target gene factor (i.e., that regulatesSRF) of miR-423-5pmuscle proliferation was obtained and diusingfferentiation TargetScan [47, 48(http://www.targetscan.org/vert_71/)], and miR-423-5p is also involved inand skeletal RNAhybrid muscle softwaredevelopment (https://bibiserv.cebitec.uni-bi and regeneration [49]. Therefore,elefeld.de/rnahybrid/) we hypothesized (Figure that MEG3 5A).and SRFSRF isare a functional crucial transcriptiontargets of miR-423-5p, factor that thatregulates is, MEG3 musclemodulates proliferationSRF andby competing differentiation for miR-423-5p.[47,48], and miR-423-5p To determine is alsothe bindinginvolved sites in skeletal between muscle miR-423-5p development and its targetand regeneration gene MEG3 [49]or SRF. Therefore,, we constructed we hypothesized wild-type that MEG3 and SRF are functional targets of miR-423-5p, that is, MEG3 modulates SRF by competing for miR-423-5p. To determine the binding sites between miR-423-5p and its target gene MEG3 or

Cells 2020, 9, 449 12 of 20

and mutant versions of MEG3 and SRF-30UTR using pGL3-Basic vectors. As shown in Figure5B, the wild-type and mutant reporter vectors contained miR-423-5p binding sites and mutated recognition sequences, respectively. Different vectors were co-transfected into PK15 cells with miR-423-5p mimic. In a dual luciferase reporter assay, we found that the luciferase activity of wild-type MEG3 and SRF remarkably decreased with the overexpression of miR-423-5p compared with the negative control groups (p < 0.01). However, no significant change was observed in the luciferase activity of the mutant group lacking miR-423-5p binding site (Figure5C,D). Interestingly, MEG3 could attenuate the decrease of luciferase activity induced by miR-423-5p in a dose-dependent manner (Figure5E). Consistently, RNA immunoprecipitation (RIP) assay using an antibody against Ago2 precipitated the Ago2 protein from our cellular extract of porcine satellite cells differentiated for 48 h. The RIP-Western blot result suggested that MEG3 had the potential to combine with miRNAs (Figure5F). The following qPCR results showed that MEG3 (Figure5G), SRF (Figure5H), and miR-423-5p (Figure5I) were significantly enriched in Ago2 pellet, confirming the interaction among MEG3, miR-423-5p and SRF. Western blot results indicated that MEG3 overexpression markedly increased SRF expression, while miR-423-5p showed opposite effect on SRF. It is worth noting that the inhibitory effect of miR-423-5p on SRF could be relieved by co-transfection with MEG3 overexpression plasmid (Figure5J). Consistent with the Western blot results in Figure3B,G, the knockdown or overexpression of MEG3 could respectively downregulate or upregulate the protein of SRF (p < 0.01). These findings confirmed the potential of MEG3 to combine with miRNAs, revealed the connection among MEG3, SRF, and miR-423-5p, and verified the sponge role of MEG3 for miR-423-5p. To further confirm the function of MEG3 as a ceRNA for miR-423-5p regulating myoblast differentiation, we performed qPCR and Western blot experiments. The results demonstrated that miR-423-5p overexpression significantly inhibited MEG3, SRF, MyoD, MyoG, and MyHC expressions at the mRNA and protein levels (Figure5K,L). Meanwhile, the immunofluorescence staining assay of MyoG and MyHC demonstrated that the number of MyoG+ and MyHC+ cells was dramatically reduced after miR-423-5p overexpression (p < 0.01); however, transfection with MEG3 overexpression plasmid could substantially relieve the inhibitory effect on myoblast differentiation (Figure5M,N). These findings verified that MEG3 regulates myoblast differentiation via abrogating the role of miR-423-5p. To verify the function of SRF, the target gene for miR-423-5p, we transfected the small interfering RNA (siRNA) of SRF or SRF overexpression plasmid into porcine satellite cells and induced differentiation for 48 h. qPCR results demonstrated that SRF knockdown remarkably downregulated MEG3 mRNA level (Figure6A). Meanwhile, mRNA and protein expression levels of MyoD, MyoG, and MyHC were considerably decreased, suggesting that si-SRF could inhibit the differentiation of porcine satellite cells (Figure6A,B). As expected, SRF overexpression considerably upregulated the expression of MEG3 and promoted the differentiation of porcine satellite cells (Figure6C,D). Collectively, MEG3 acts as a ceRNA for miR-423-5p to attenuate the inhibitory effect on SRF, thereby promoting the differentiation of porcine satellite cells (Figure7). Cells 2020Cells, 20209, 449, 9, x 13 of 2013 of 20

Figure 5. MEG3 is a target of miR-423-5p during myoblast differentiation. (A) The miR-423-5p target

sequenceFigure on 5. MEG3 isand a target 30 UTR of miR-423-5p of SRF. The during red letters myoblast represent differentiation. the seed (A region) The miR-423-5p of miR-423-5p target and its targetsequence region. on The MEG3 underlined and 3′ UTR word of SRF letters. The in red red letters show represent the mutated the seed site. region (B) Theof miR-423-5p sequencing and results its of

MEG3targetand region.SRF-3 0TheUTR underlined wild and word mutant letters types. in red The show red the boxes mutated represent site. ( theB) The mutant sequencing bases. results (C) Luciferase of activitiesMEG3 assay and inSRF PK15-3′ UTR cells wild were and measured mutant aftertypes. transfection The red boxes for 48represent h with MEG3the mutantwild bases. or mutant (C) type pGL3-BasicLuciferase plasmids activities togetherassay in PK15 with cells miR-423-5p were measured mimic. after (D transfection) Luciferase for activities 48 h with assay MEG3 in wild PK15 or cells mutant type pGL3-Basic plasmids together with miR-423-5p mimic. (D) Luciferase activities assay in were measured after transfection of 48 h with SRF wild or mutant type pGL3-Basic plasmid together PK15 cells were measured after transfection of 48 h with SRF wild or mutant type pGL3-Basic with miR-423-5p mimic. (E) Wild type of MEG3 fragments rescued the relative luciferase activities of plasmid together with miR-423-5p mimic. (E) Wild type of MEG3 fragments rescued the relative SRF wild-type pGL3-Basic plasmid in a dose-dependent manner. (F–I) RIP assay was conducted with luciferase activities of SRF wild-type pGL3-Basic plasmid in a dose-dependent manner. (F–I) RIP Ago2assayantibody. was conducted Protein level with wasAgo2 detected antibody. by Protein IP-Western level was Blot detected (F). MEG3 by IP-Western(G), SRF ( HBlot), and(F). MEG3 miR-423-5p (I) were(G), SRF detected (H), and by qPCR.miR-423-5p (J) The (I) were protein detected expression by qPCR. level (J) ofTheSRF proteinshowing expressionMEG3 leveloverexpression of SRF increasesshowingSRF MEG3expression overexpression and relieves increases the inhibitory SRF expression effect induced and relieves by miR-423-5p the inhibitory on effectSRF.( inducedK,L) Relative mRNAby miR-423-5p (K) and protein on SRF expression. (K,L) Relative level mRNA (L) changes(K) and protein of MEG3, expression SRF, and level the (L) dichangesfferentiation of MEG3, marker genesSRF, after and transfection the differentiation with miR-423-5pmarker genes mimic after transfection or mimic NC. with ( MmiR-423-,N) Immunofluorescence5p mimic or mimic NC. staining of MyoG(M,N)( MImmunofluorescence) and MyHC (N) in staining porcine of satellite MyoG cells(M) diandfferentiated MyHC (N for) in 48 porcine h after satellite transfection cells with miR-423-5pdifferentiated mimic, forMEG3 48 h+ aftermiR-423-5p transfection mimic, with or correspondingmiR-423-5p mimic, NC. MEG3 Statistical+miR-423-5p results ofmimic, the positively or stainedcorresponding cells show NC. that miR-423-5pStatistical results significantly of the po reducessitively the stained percentage cells show of MyoG thatand miR-423-5pMyHC, while significantly reduces the percentage of MyoG and MyHC, while the co-transfection with MEG3 can the co-transfection with MEG3 can recover their reduction. Scale bar of (M): 50 µm. Scale bar of recover their reduction. Scale bar of (M): 50μm. Scale bar of (N): 100μm. The numbers below the (N): 100 µm. The numbers below the Western blots (J) mean the fold change of SRF protein quantities Western blots (J) mean the fold change of SRF protein quantities related to the mimic NC group. The relatedrelative to the mRNA mimic levels NC were group. normalized The relative to those mRNA of the levelscontrol were 18S rRNA normalized. The relative to those protein of thelevels control 18S rRNA. The relative protein levels were normalized to those of the control β-tubulin. Error bars represent mean SEM of three biological replicates. Statistical significance of differences was assessed ± by Student’s t-test. * p < 0.05, ** p < 0.01. NC, negative control. UTR, untranslated regions. RIP, RNA immunoprecipitation. qPCR, quantitative polymerase chain reaction. Cells 2020, 9, x 14 of 20

were normalized to those of the control β-tubulin. Error bars represent mean ± SEM of three biological replicates. Statistical significance of differences was assessed by Student’s t-test. * p < 0.05, ** p < 0.01. NC, negative control. UTR, untranslated regions. RIP, RNA immunoprecipitation. qPCR, quantitative polymerase chain reaction.

To verify the function of SRF, the target gene for miR-423-5p, we transfected the small interfering RNA (siRNA) of SRF or SRF overexpression plasmid into porcine satellite cells and induced differentiation for 48 h. qPCR results demonstrated that SRF knockdown remarkably downregulated MEG3 mRNA level (Figure 6A). Meanwhile, mRNA and protein expression levels of MyoD, MyoG, and MyHC were considerably decreased, suggesting that si-SRF could inhibit the differentiation of porcine satellite cells (Figure 6A,B). As expected, SRF overexpression considerably upregulated the expression of MEG3 and promoted the differentiation of porcine satellite cells (Figure 6C,D). Collectively, MEG3 acts as a ceRNA for miR-423-5p to attenuate the inhibitory effect Cells 2020, 9, 449 14 of 20 on SRF, thereby promoting the differentiation of porcine satellite cells (Figure 7).

Figure 6. SRF promotes the differentiation of porcine satellite cells. (A) qPCR results showing that SRF knockdown significantly decreases the expression levels of MEG3, MyoD, MyoG, and MyHC in in Figure 6. SRF promotes the differentiation of porcine satellite cells. (A) qPCR results showing that porcine satellite cells differentiated for 48 h. (B) Western blot analysis results showing SRF knockdown SRF knockdown significantly decreases the expression levels of MEG3, MyoD, MyoG, and MyHC in significantly decreases the protein expression of SRF and the differentiation marker genes. (C,D) qPCR (inC )porcine and Western satellite blot cells (D) resultsdifferentiated showing forSRF 48overexpression h. (B) Western significantly blot analysis increases results theshowing expression SRF levelsknockdown of MEG3 significantlyand promotes decreases porcine the satellite protein cell expression differentiation. of SRF Theand relativethe differentiation mRNA levels marker were normalizedgenes. (C,D to) qPCR those of(C the) and control Western18S rRNA blot. The(D) relativeresults proteinshowing levels SRF were overexpression normalized to significantly those of the controlincreasesβ-tubulin the expression. Error bars levels represent of MEG3 mean andSEM promotes of three biologicalporcine satellite replicates. cell Statistical differentiation. significance The relative mRNA levels were normalized to those± of the control 18S rRNA. The relative protein levels Cells 2020of di, 9ff, xerences was assessed by Student’s t-test. * p < 0.05, ** p < 0.01. NC, negative control. qPCR,15 of 20 quantitativewere normalized real-time to those polymerase of the chain control reaction. β-tubulin. Error bars represent mean ± SEM of three biological replicates. Statistical significance of differences was assessed by Student’s t-test. * p < 0.05, ** p < 0.01. NC, negative control. qPCR, quantitative real-time polymerase chain reaction.

Figure 7. Proposed model of MEG3 regulatory mechanism in porcine satellite cell differentiation. In this Figuremodel, 7.MEG3 Proposed, as a ceRNA,model of promotes MEG3 regulatory porcine satellite mechanism cell di inff erentiationporcine satellite by sponging cell differentiation. miR-423-5p In to thisrelieve model, the inhibiting MEG3, easff ecta onceRNA,SRF. promotes porcine satellite cell differentiation by sponging miR-423-5p to relieve the inhibiting effect on SRF. 4. Discussion 4. DiscussionIn this study, we demonstrated that MEG3 acted as a key regulator in myogenesis and revealed a novelIn molecular this study, mechanism we demonstrated by which thatMEG3 MEG3regulated acted theas a miR-423-5p–SRF key regulator inaxis. myogenesis Our results and suggested revealed athat novelMEG3 molecular, as a ceRNA, mechanism promotes by the which differentiation MEG3 regulated of porcine the satellite miR-423-5p–SRF cells by sponging axis. Our miR-423-5p results suggestedto relieve thethat inhibiting MEG3, as e ffa ectceRNA, on SRF promotes(Figure 7the). differentiation of porcine satellite cells by sponging miR-423-5pProliferation to relieve and ditheff erentiationinhibiting effect of myoblasts on SRF are (Figure crucial 7). processes for skeletal muscle development, whichProliferation determine theand quality differentiation and quantity of ofmyoblasts agricultural are animalcrucial meat processes production. for skeletal Elucidating muscle the development, which determine the quality and quantity of agricultural animal meat production. Elucidating the regulatory mechanisms of myogenesis helps to find therapeutic targets for muscle disease and improve meat traits in animal production. Therefore, understanding the underlying mechanisms of myogenesis is particularly important. Although thousands of lncRNAs have been identified [38,50], a minority of functional lncRNAs, such as TncRNA [51], lncMD [52], H19 [52–56], and lncIRS1 [57], are involved in mammal myogenesis. MEG3, a differentially expressed lncRNA during postnatal skeletal muscle development in pigs, has four crucial polymorphism sites associated with back fat thickness [39]. Li et al. first confirmed the two overlapping fragment isoforms of MEG3 with respective lengths of 1160 and 1219 bp in Yorkshire and Korean native pigs [27]. Our RACE results confirmed the two variants in pigs and showed that they were 1430 and 1380 bp in full length. Subsequently, the tissue expression profile showed the advantage of their expression and the high abundance of MEG3 v2 in skeletal muscle tissues, which was consistent with the results of a previous study in various tissues and six developmental stages of longissimus dorsi muscle during porcine postnatal development [58]. In addition, the high expression level of MEG3 during the early stage of myogenesis in pigs and cattle collectively indicated its important role in myogenesis and muscle development [35]. Our research results demonstrated that MEG3 knockdown remarkably decreased the expression of myogenic marker genes in mRNA and protein levels; however, the overexpression results of the two transcripts of MEG3 were opposite. The findings conceivably indicated that MEG3 could act as an accelerator in porcine myogenic differentiation, which is also consistent with a previous study in cattle [35]. Furthermore, we further assessed differential gene expression after MEG3 knockdown, with differentiation for 30 and 40 h, and provided global insights into gene functions during myogenesis using an RNA-seq approach. KEGG pathway and GO term enrichment analysis found the involvement in muscle-related processes, such as Z disc, actin cytoskeleton, myofibril, muscle contraction, skeletal muscle cell

Cells 2020, 9, 449 15 of 20 regulatory mechanisms of myogenesis helps to find therapeutic targets for muscle disease and improve meat traits in animal production. Therefore, understanding the underlying mechanisms of myogenesis is particularly important. Although thousands of lncRNAs have been identified [38,50], a minority of functional lncRNAs, such as TncRNA [51], lncMD [52], H19 [52–56], and lncIRS1 [57], are involved in mammal myogenesis. MEG3, a differentially expressed lncRNA during postnatal skeletal muscle development in pigs, has four crucial polymorphism sites associated with back fat thickness [39]. Li et al. first confirmed the two overlapping fragment isoforms of MEG3 with respective lengths of 1160 and 1219 bp in Yorkshire and Korean native pigs [27]. Our RACE results confirmed the two variants in pigs and showed that they were 1430 and 1380 bp in full length. Subsequently, the tissue expression profile showed the advantage of their expression and the high abundance of MEG3 v2 in skeletal muscle tissues, which was consistent with the results of a previous study in various tissues and six developmental stages of longissimus dorsi muscle during porcine postnatal development [58]. In addition, the high expression level of MEG3 during the early stage of myogenesis in pigs and cattle collectively indicated its important role in myogenesis and muscle development [35]. Our research results demonstrated that MEG3 knockdown remarkably decreased the expression of myogenic marker genes in mRNA and protein levels; however, the overexpression results of the two transcripts of MEG3 were opposite. The findings conceivably indicated that MEG3 could act as an accelerator in porcine myogenic differentiation, which is also consistent with a previous study in cattle [35]. Furthermore, we further assessed differential gene expression after MEG3 knockdown, with differentiation for 30 and 40 h, and provided global insights into gene functions during myogenesis using an RNA-seq approach. KEGG pathway and GO term enrichment analysis found the involvement in muscle-related processes, such as Z disc, actin cytoskeleton, myofibril, muscle contraction, skeletal muscle cell differentiation, and structural constituent of muscle. As a consequence, it is reasonable to infer that MEG3 plays an important role in regulating porcine skeletal muscle development. To reveal the underlying molecular mechanism of MEG3, we explored a large number of existing studies. We found that several functional lncRNAs have been characterized, and their function can be affected by multiple mechanisms. Partial lncRNAs, such as the new lncRNA SYISL [25], lncRNA Mata1 [59], and Linc-YY1 [24], regulate myoblast differentiation and skeletal muscle regeneration by recruiting chromosome modification complexes to the promoters of target genes. Other lncRNAs, such as Linc-RAM [40] and Myoparr, can recruit transcription factor MyoD and RNA-binding protein complex Ddx17/PCAF, respectively, to myogenic marker gene promoters to further promote myogenic differentiation and regeneration [60]. Also, LncMyoD acts as a competitive binding regulator to attenuate the binding ability of IMP2 for its target genes and inhibit myoblast differentiation [22]. Notably, subcellular localization determines the regulatory mechanism. Many cytoplasm-located lncRNAs can act as a ceRNA to sponge miRNA and relieve the inhibitory effect on target genes. Muscle-specific lncRNA, Linc-MD1, serves as a molecular sponge of miR-133 and miR-135 to relieve the repression of MAML1 and MEF2C and induce skeletal muscle differentiation [19]. Overexpression of lncRNA MAR1 can promote myogenic differentiation by effectively weakening the inhibitory effects of miR-487b on Wnt5a [61]. MEG3 is mainly found in the nucleus of porcine satellite cells, and very rarely is located in the cytoplasm [33,62]. However, the proportion of MEG3 in the cytoplasm was remarkably increased in the myotube in the present study. This result led us to hypothesize that MEG3 may act as a ceRNA to regulate the differentiation of porcine satellite cells similar to cytoplasm-located lncRNAs. Similarly, miR-9 can regulate the expression of nuclear lncRNA MALAT1 by directly binding with miRNA recognition elements and in an Argonaute-2-dependent manner in human L428 and U87MG cells [63]. In addition, MALAT1 is translocated from the nucleus into the cytoplasm during the G2/M cell cycle phase by interacting with heterogenous nuclear RNP C in the cytoplasm [64], where MALAT1 acts as a ceRNA for miR-133 and modulates SRF to promote the differentiation of C2C12 cell line [59]. This may be a reasonable explanation for the increased proportion of MEG3 in the cytoplasm from primary porcine myoblast to myotube in the present study. Therefore, we further affirmed the molecular sponge role of MEG3. Cells 2020, 9, 449 16 of 20

Software prediction analysis revealed that MEG3 certainly shared the same miRNA recognition sites for miR-423-5p with SRF, a crucial transcription factor for myogenesis process. Previous research showed functional SRF is required for the differentiation of C2C12 cells and the regulation of MyoD expression [47]. Deletion of SRF severely suppresses the muscle formation of muscle progenitors in mammalian embryonic development process [48], blocks cell fusion, and inhibits the synthesis of MyoD, MyoG, and MyHC, exerting severe muscle atrophy [47,65]. Similarly, SRF mutant mice died from severe skeletal muscle myopathy characterized by a deficiency in muscle growth during the perinatal period by inhibiting the recruitment of myocardin-related transcription factors [48,66,67]. Our observations in SRF siRNA and overexpression group provided evidence on the role of SRF in myogenesis. In addition, we found that SRF had conservative complementary sites with miR-423-5p, which is a potential regulator of myogenesis and plays a negative role during myoblast proliferation and differentiation by targeting the suppressor of fused homolog [49]. Because the Ago-RIP method described in Werfel et al. had been performed to seek ceRNA for a specific miRNA [68], we carried out an RNA immunoprecipitation assay using antibody against Ago2. The RIP-Western blot result showed the potential of MEG3 to combine with miRNAs. The enrichment of MEG3, SRF and miR-423-5p in Ago2 pellet revealed the connection among them. Subsequently, luciferase activity assays verified that MEG3 acted as a molecular sponge to adsorb miR-423-5p. MEG3 overexpression could effectively recover the reduced luciferase activity of wild-type MEG3 and SRF induced by miR-423-5p. Consistently, the inhibitory effect of miR-423-5p on SRF and myoblast differentiation could be abolished by MEG3 overexpression. These findings verified the novel functional mechanism that MEG3 acts as a ceRNA to sponge miR-423-5p, which weakens the suppression on SRF, thereby promoting the differentiation of porcine satellite cells. In addition, the fact that MEG3 was detected mainly in the nucleus implies that it can regulate myogenesis though other mechanisms, which remain to be further explored. In conclusion, MEG3 is a vital regulator that inhibits myoblast proliferation and promotes myoblast differentiation in porcine satellite cells. Our findings suggested the novel functional mechanism of MEG3, which acts as a molecular sponge of miR-423-5p to upregulate the target gene SRF expression level during the differentiation of porcine satellite cells. Our research provides new insights into the molecular mechanisms of MEG3 in porcine myogenesis and contributes to a better understanding of molecular regulation of lncRNA in multiple pathways.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/2/449/s1, Supplementary Table S1: Oligonucleotide sequences in this study, Supplementary Table S2: Information of Primers, Supplementary Table S3: Summary of RNA-seq data. Author Contributions: Conceptualization, X.C. and C.L.; Formal analysis, X.C. and L.L.; Funding acquisition, C.L.; Investigation, M.L.; Methodology, X.C., G.S. and L.C.; Project administration, C.L.; Resources, C.L.; Supervision, C.L. and C.F.; Visualization, X.C., G.S. and L.C.; Writing—original draft, X.C.; Writing—review & editing, X.C., C.F. and C.L. All authors read and approved the final manuscript. Funding: The work was supported by National Natural Science Foundation of China under Grant No.31872322; the Fundamental Research Funds for the Central Universities under Grant No.2662017PY030; National Key Project of Transgenic Animal and Crops of Ministry of Science and Technology of China under Grant No. 2016ZX08006-003. Acknowledgments: The authors acknowledge Jinlong Zhang for the help on piglet management and feeding. The authors acknowledge Wenzhe Luo, Jingxun Li, and Sheng Wang for their guidance and assistance on the isolation of porcine satellite cells. Conflicts of Interest: We declare that there are no competing interests.

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